Over the next year it is planned to attempt to purify the acid-resistant nucleational factor in cartilage micropuncture fluid (Cfl). A new technique of band centrifugation and of microcolumn chromotography whereby cartilage fluid proteoglycans can be separated on Sepharose 2B into aggregate and subunit forms has already been developed. The site on these chromatographic systems where alkaline phosphatase and the nucleational factor appear have been determined and run on SDS capillary gels. The nucleational factor will be studied by precipitation in an in vitro model system of calcium and phosphate from a synthetic lymph. The calcium will be measured by our new helium glow photometric method for calcium. The purified proteoglycan aggregates will be dissociated and the nature of the link glycoprotein studied to see if it could be an alkaline phosphatase or a calcium-binding nucleating factor. Further studies for the presence of membranes or membrane fragments of matrix vesicles will be done in the Cfl and their relationships to nucleational factor determined. Further studies on the effect of highly purified lysozyme on proteoglycan aggregates isolated from Cfl will be done to obtain proof of previously postulated mechanism of Cfl proteoglycan dissociation and initiation of mineral formation of Cfl. When a nucleational factor is isolated either from the proteoglycan aggregates or other fractions, these will be studied for their biochemical properties, and presence on cartilage collagen or in matrix vesicles sought. BIBLIOGRAPHIC REFERENCES: Howell, D.S.: Calcification Mechanisms (Bone Formation). Isr. J. Med. Sci. 12:3-9, 1976. Howell, D.S., Pita, J.C. and Alvarez, J.: Possible Role of Extracellular Matrix Vesicles in Initial Calcification of Healing Rachitic Cartilage. Fed. Proc. 35:122-126. 1976.